The public health impact of substance abuse is enormous and widespread; adverse consequences include mortality, morbidity including debilitating physical and psychological symptoms, and loss of productivity. Existing drugs are not completely effective and often characterized by adverse side effects; hence, there is a great need to discover new neurochemical targets for the development of addiction therapeutics. As a neurochemical class, peptides are understudied relative to classic neuromodulators such as dopamine. Yet, work on a relatively small number of a priori-identified peptides show that they play profound functional roles across the addiction cycle. The next generation of anti-addiction medications could very conceivably be based on yet undiscovered peptides with specific functional roles in the addiction cycle. In this proposal, we aim to apply a cutting-edge mass spectrometry (MS)-based analytic platform to discover new peptides (peptidomic discovery), and characterize already-known peptides with unprecedented precision, across two drug states: acute cocaine intoxication and peak cocaine withdrawal. The technological platform consists of the following methods: 1. Shotgun peptidomics approach based on gas-phase tandem MS fragmentation methods coupled with isobaric tagging to rapidly quantify a large number of neuropeptides; 2. Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) of thin tissue slices using a novel gold-nanoparticle matrix, in correlation with in situ hybridization, to map the spatial distribution of peptide fluxes and enable co-localization wih dopamine signals; 3. Affinity-enhanced microdialysis using innovative magnetic nanoparticles to massively amplify the recovery of peptide efflux from the central nervous system (CNS) extracellular space, thereby improving sensitivity and temporal resolution; 4. Synthesis of any novel peptides discovered, and behavioral testing of these sequences in a well- validated brain stimulation-reward threshold procedure. These methods are highly advanced, and some new innovations have never been tried before in mammalian tissues. Hence, considering the time constraints of the R21 grant mechanism, our first goal is to refine and optimize the technological platform by specifically focusing on simultaneous ?-opioid, ?-opioid, and dopamine co-transmission in the nucleus accumbens (Acb) and prefrontal cortex (PFC) during acute cocaine intoxication and peak cocaine withdrawal. This type of combinatorial analysis in both brain sites has never been attempted. Our work has the potential to reveal crucial insights regarding an important opponent-process theory in addiction biology, positing diverse and opposing ?- opioid actions relative to ?-opioid/dopamine actions across the addiction cycle. Choosing acute cocaine intoxication and peak withdrawal offers the strongest test of the theory. Insights gained from these analyses could translate into uniquely effective combinatorial treatment strategies. Our second goal is to test any new peptide sequences discovered in the MS-based shotgun peptidomic analysis in the subsequent stages of our platform, as time permits. This plan will allow us to refine a powerful, technologically advanced platform for peptidomic discovery, and achieve unprecedented 'vertical integration' from the transcriptional to the behavioral levels. At the same time, our studies will ask a discrete question of great scientific importance regarding the interplay among opioid peptides and dopamine in distinct cocaine-associated states.